Rate-limiting Steps in theTension Development of Freeze-glycerinatedVascular Smooth Muscle

JOHN W. PETERSON IIIPhysiologisches Institut, Universitat Heidelberg, D-6900 Heidelberg 1, Federal Republic ofGermany

ABSTRACT A method for "skinning" arterial smooth muscle is presentedwhich yields isometric tension development typically 60-80% of maximumphysiological tension in the presence of micromolar Ca" and millimolar Mg-ATP, while retaining essentially the native protein content . Using the methodsof "Ca jump," the time-course of Ca"-activated tension development in theskinned artery can be made identical to, but not faster than, the rate of tensiondevelopment in the intact artery . In the skinned artery, activating free [Ca"]does not substantially alter the rate at which tension development approachesthe final steady tension attained at that free [Ca"] (<25% decline in speed fora 10-fold decrease in [Ca"]) . These observations are taken to mean that therate-limiting step in isometric tension development in arterial smooth muscledoes not depend directly on Ca".

INTRODUCTION

Much work has recently appeared using preparations of "skinned" smoothmuscle to study the processes of activation and contraction (Sparrow et al .,1981 ; Kerrick et al ., 1980; Peterson, 1980 ; Mrwa et al ., 1979; Gordon, 1978;Endo et al ., 1977) . Skinning processes are primarily methods by whichmembranes are rendered highly permeable so that the intracellular milieu canbe controlled by altering the extracellular environment, while nonethelessleaving the contractile apparatus functional . Data obtained from such systemsthat retain contractility therefore lie midway between studies on isolatedcontractile proteins and studies on physiologically intact smooth muscle .To obtain fair comparison between studies on skinned and intact smooth

muscle, it should be established that the contractile properties of the skinnedpreparations are at least comparable to those of the intact preparations . Inmany studies in the literature, this has not been done. In this report, I describea skinning method that renders the smooth muscle cells of hog carotid arteryhighly permeable to low molecular weight substances, but retains essentiallythe contractile properties and native protein content of the intact artery .

Address correspondence to Dr . John W. Peterson III, Massachusetts General Hospital, Neuro-surgical Service, Warren Building 465, Boston, Mass . 02114.

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438 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 " 1982

Maximum isometric tension development of the skinned artery segments inmicromolar Ca" and millimolar Mg-ATP ranges from 60 to 80% of theobserved maximum physiological tension development .

Studies on isolated smooth muscle proteins to identify the rate-limitingsteps in the activation process have been equivocal . Mrwa and Hartshorne(1980) have reported that whereas isolated myosin light chains are phospho-rylated much more rapidly than the actomyosin cross-bridge cycling rate,light chains attached to whole myosin (a situation more similar to the in vivocase) are phosphorylated at about the same rate or slower than the cross-bridge cycle rate . This allows the possibility that phosphorylation is the rate-limiting step . A mathematical model of this possibility has recently beenpresented and verified for the case of skinned guinea pig taenia coli smoothmuscle (Peterson, 1982b) . Driska et al . (1981), on the other hand, have shownin the intact hog carotid artery that myosin light chain phosphorylationproceeds more rapidly than tension development, which suggests that othersteps are rate limiting.

In these studies with skinned vascular smooth muscle, by the use ofappropriate solutions designed to minimize rate limitations due to the inwarddiffusion of activating Ca", the time-course of isometric tension developmentcan be made identical to that of the physiologically intact artery, but notfaster . This suggests that the in vivo rate-limiting step in tension developmentis not directly dependent on Ca"; that is, some step after Ca" activation israte limiting for tension development in vascular smooth muscle .

MATERIALS AND METHODS

In earlier work, the glycerination method used successfully with skeletal muscles(prolonged soaking in cold glycerol solutions) gave contractile responses in smoothmuscles only 5-10% of maximum physiological tension development (Filo et al ., 1965 ;Mrwa et al ., 1974) . A modification of this method ("freeze-glycerination") givescontractile responses 40-80% of full physiological tension development in a variety ofsmooth muscles upon application of micromolar Ca" in the presence of millimolarMg-ATP (Peterson, 1980) .

Tissue Preparation

Hog carotid arteries were collected at an abattoir within 15 min of animal death,cleaned of blood and loose adventitia, and stored in a cold or room temperaturephysiological saline solution (PSS) . Small segments of artery were prepared by firstdissecting out the smooth muscle-containing media layer following the methoddescribed by Gluck and Paul (1977), laying the media strip out flat on Parafilm, andslicing off thin (0.1-0.3 mm) transverse sections in the direction of smooth muscleorientation with a razor blade . In tests of physiological contractility, this methodproved more reliable than "pulling out" media strips, which appears to overstretchthe tissue and leads to reduced tension development (cf. Driska et al ., 1981) . Thismethod produces artery segments typically 8mm long with a rectangular cross-section^-0.1-0.3 mm (as desired) by 0.6-0.8 mm with a wet weight of 1-2 mg. Forcomparative studies, ^"20 pieces per artery were prepared and allowed to equilibratein oxygenated PSS at 37°C for 1 h . The pieces were then gently drained of PSS,immersed in 5 ml of cool (5 ° C) glycerinating solution, and allowed 10 min to

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equilibrate . The beaker was then placed in a freezer at -25 °C . Artery segments wereleft in the freezer until needed, then rinsed with relaxing solution and mounted in anisometric tension apparatus . Within - 15 min in the freezer, physiological contractility(i .e ., responsiveness to normal agonists such as norepinephrine, histamine, or highK') was completely abolished and isometric tension development depended whollyon externally provided Ca" and Mg-ATP . Tissues stored frozen as long as 1 yrshowed no deterioration in properties such as absolute force generation or the force[Ca"] relation .

Permeability (as measured by the equilibration rate with [ 3H]ATP) is high : DATP^-2 X 10-s cm2/s . Due to the nature ofthe method, this high permeability is probablydue to extensive membrane cracking rather than wholesale membrane dissolution .That the freezing (or thawing) step produces the permeability increase is evidencedby the fact that artery segments tested after the 5-10 min equilibration soak inglycerinating solution showed normal physiological contractility and no contractileresponse to millimolar Mg-ATP and micromolar Ca". Preservation of some mem-brane structure for force transmission, better retention of native protein content, andthe fact that the "skinning" occurs homogeneously throughout the preparation ratherthan progressively (as in detergent treatments, which can require 16 h at roomtemperature to be effective ; Gordon, 1978) perhaps account for the superior tensiondevelopment with this skinning method.

SolutionsBecause the stability of isometric tension in the skinned artery segments was found tobe very sensitive to high ionic strength, solutions were prepared to maintain ionicstrength as low as feasible and always <0.1 M. The relaxing solution contained 20mM imidazole, 5 mM EGTA, 5 mM Na2ATP, 5 mM MgC12 and, as ATP-regeneratingsystem, 5 mM K-phosphoenolpyruvate with 20 U/ml pyruvate kinase (Sigma typeIII ; Sigma Chemical Co., St . Louis, Mo.) . Solutions were titrated with KOH to pH6.85 to 7.0 as desired at 37°C and KCl was added to a final ionic strength of 0.085 M.Under these conditions, Mg` binding to ATP and phosphoenolpyruvate provides areasonably well-buffered free [Mg"] of ^-0.4 mM. Activating solutions containedadded CaC12 with reduced KCl and increased KOH, so that ionic components (exceptfor Ca-EGTA and free Ca") were virtually identical to relaxing solution . Free Ca"concentration (pCa = -log[Ca++]) and other ionic components were computed fromthe data of White and Thorson (1972) . In the "Cajump" relaxing solution, EGTAwas reduced to 0.1-0.2 mM and the ionic strength was compensated by the additionof HDTA (1,6-diamino-hexan-n,n,n ,n'-tetraacetic acid ; Fluka AG, Switzerland) to aconstant tetraacid concentration of 5 mM (Moisescu, 1976) .The freeze-glycerinatinn solution contained 50% relaxing solution ; 50% glycerol

(vol/vol) with 2 mM dithiothreitol added .

Tension MeasurementsThe small skinned smooth muscle segments were attached horizontally between afixed, bent glass rod and a thin carbon filament-epoxy extension rod from an AE 801force transducer (Aksjeselskapet Mikroelektronikk, Horten, Norway), using a smallamount of fast water-setting cyanoacrylate glue . System compliance amounted to<0.2% shortening at maximum isometric force (typically 10-20 mN) and the trans-ducer speed exceeded 8 kHz . Tissue segments were mounted in relaxing solution,stretched to a peak force ^-10 mN, and allowed to stress relax. This cycle of stretchand relaxation was repeated twice (-20 min) and the tissue length was then adjusted

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to a passive force of - 1 mN. Tissues were incubated in 0.9-ml glass cups of theappropriate solutions, which were rotated at -1 Hz to provide stirring and thermo-stated at 37 °C (tl ° ) . The cross-section area was computed from the segment wetweight/length .

Diffusion Measurements

THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 " 1982

Small segments of arteries were preweighed and mounted for force measurements asdescribed . Tissues were then incubated for 8 min in relaxing solution (pCa > 8)without ATP and then for 10 min in ATP-free contracting solution (pCa 6.4) ; ionicstrength was maintained with added KCI and free [Mg"] with MgC12 . In the absenceof ATP, Ca" produced no contraction . At time t = 0, 2 mM Mg-ATP with [2,8_ 3H]ATP at specific activity 0.05 Ci/mmol or trace amounts of [3H(G)]inulin were added(New England Nuclear, Boston, Mass.), mixed, and an aliquot of the bathing solutionwas taken . At various times after the onset of tension development, the bathingsolutions were again sampled and tissues were removed from the labeled ATPsolutions, gently blotted to remove adherent surface solution, weighed, and dissolvedin 0.5 ml Soluene-350 (Packard Instrument Co., Inc ., Downers Grove, 111 .) at 50°C .Two aliquots of the tissue extract and the incubating solutions were then counted in5 ml Dimilume (Packard Instrument Co., Inc .) . The ratio of average counts per unitvolume in the artery piece (assuming density = 1 .0) to average counts per unit volumein the incubating solution was used as an index of equilibration rate .

Tissue ATPase MeasurementsBecause of the ATP-regenerating system used in the incubating solutions, the totalATPase of the skinned artery segment appears as accumulation of pyruvate (ADP +phosphoenolpyruvate --> ATP + pyruvate with pyruvate kinase) . In control experi-ments, the artery segments metabolized no pyruvate added directly to the incubatingsolutions in the absence of added NADH. In each ATPase measurement, two 0.9-mlincubation cups were filled with appropriate solutions ; one cup contained the tissueand the other served as control for spontaneous decomposition of ATP and phospho-enolpyruvate . After a chosen time interval (usually 10 min), both solution cups werebrought to 0.25 mM NADH, 37.5 mM Tris, 7.5 mM MgC12 at pH 8.6 by the additionof 15 IAI concentrate . The difference in absorbance change between the two cups at340 nm upon the addition of 10 Al lactate dehydrogenase (Sigma type II) was equatedto the ATP hydrolyzed using a calibration factor 6 (t0.2) A34o units/mM ADP.

Protein ContentThe total protein content of various solutions and tissue extracts were determined bythe biuret method with bovine serum albumin as standard . Protein was extracted byhomogenization and continual stirring for 12-16 h at 4°C in 80 mM KCI, 20 mMimidazole, 5 mM EGTA, 10 mM ATP, and 1 mM dithiothreitol (pH 7.2), followedby centrifugation at 20,000 g for 15 min .

RESULTS

Protein Content

A comparison was made between intact and "freeze-glycerinated" arterieswith regard to contractile protein content both by protein assay and sodiumdodecyl sulfate (SDS) polyacrylamide gel electrophoresis . Artery segmentswere prepared as described in Methods, some freeze-glycerinated and others

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left intact in PSS. Several small pieces of each (typically 4-6 mg) were finelyminced and incubated with shaking for 0.5 h at 50°C in 1% SDS, 1%mercaptoethanol, 20 mM imidazole, and 5 mM EGTA (pH 6.85) ; they werethen briefly centrifuged, and the supernatant was applied to 6% polyacryl-amide gels (0 .1 M phosphate, 0.1% SDS, pH 7) . The resulting gels showed amultitude ofpeaks (-40) . In repeated trials, visual comparison ofdensitometertracings gave no substantial differences between the glycerinated and livingsamples.

Similarly, larger samples of artery (typically 2-3 g) were freeze-glycerinatedor left intact, and treated with conditions used to extract crude actomyosin(cf. Russell, 1973) . The extractable protein content of the intact arteriesaveraged 46.8 (± 4.3 SD, n = 4) mg protein/g wet tissue, which is comparableto the value determined by Cohen and Murphy (1978) for the content of actin+ myosin + tropomyosin in hog carotid artery (43 ± 5 mg protein/g wettissue) . Batches of freeze-glycerinated arteries were extracted under identicalconditions, giving an average extractable protein content of 49.2 (± 8 .9 SD,n = 3) mg protein/g wet tissue . Gel densitometry of the crude extract run onSDS polyacrylamide gels as above (Fig . 1) shows that the contractile proteinsconstitute the bulk of the protein extractable from freeze-glycerinated arteries .The two very prominent bands at ^-32,000 and 23,000 mol wt are notidentified, but can be greatly reduced by precipitation of the crude actomyosinby dialysis against ATP-free buffer.

Differences in important proteins of a regulatory nature present in lowconcentrations would not be detected by the above methods. To test freeze-glycerinated arteries for protein elution under the experimental conditions, alarge batch of skinned artery segments (^ "4 g) were incubated with gentleshaking for 1 h at 37°C in 2 ml of normal relaxing solution, which was thenassayed for protein content . The value determined was <0.5 mg/ml, whichindicates that -0.5% of extractable protein is eluted from the skinned arterysegments during a typical experimental time period .

Dependence of Tension Development on [Ca"]Fig. 2 shows the average measured dependence of stable isometric tensiondevelopment in nine skinned artery segments from five arteries as a functionof free [Ca"], where the data have been grouped according to pCa andaveraged as illustrated by the standard error bars shown . Maximum Ca"-activated force (pCa 4.5) at 5 mM Mg-ATP was typically 8-9 mN, or -r60%of the maximum force measured in identically prepared artery segments thatwere not freeze-glycerinated and stimulated with high K+ and 10-5 Mhistamine (cf. Peterson, 1982). In individual experiments, however, tensionsas high as 90% of maximum physiological tension were occasionally observed .The sigmoidal shape of the force [Ca++ ] relation is as expected for a musclepreparation and is well fit by the functional form used for cooperative bind-ing models, 1/(1 + (K/[Ca++])N) . The observed value of N, which determinesthe steepness of the relation, was estimated from a linearized least-squares fitand is not substantially different from that reported for other skinned smooth

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muscle preparations . Gordon (1978) found a value of ^-3 for detergent-skinnedrabbit taenia coli, whereas the data of Filo et al . (1965) indicate a value ^ " 2.5for hog carotid artery. The value of K, which in some sense represents anapparent binding constant for activating Ca" (cf. Edsall and Wyman, 1958),is shifted substantially to the left relative to other smooth muscles. Data fromvisceral smooth muscles (Gordon, 1978 ; Endo et al ., 1977) indicate that thevascular preparation is some four- to fivefold more Ca" sensitive. In a directcomparison, Endo et al . (1977) observed a similar difference in pK for theforce [Ca"] relations measured in rabbit taenia coli and pulmonary artery .

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Densitometer tracing of the protein extracted from freeze-glyceri-nated arteries and subjected to SDS polyacrylamide gel electrophoresis andstained with Coomassie Blue . The molecular weight scale shown (X 10 3 mol wt)was obtained from calibrations using cross-linked hemoglobin and cross-linkedalbumin (Sigma Chemical Co.) .

The saponin-treatment used there, however, led to a rapid decline in maxi-mum Ca"-activated tension development upon repeated contractions . Max-imum tension development in the freeze-glycerinated artery preparation, onthe other hand, was usually reproducible to better than 10% with repeatedCa++ activation . Long-term incubation at 37°C, however, led to a failure torelax completely upon subsequent Ca" removal . The force [Ca"] relationhere is somewhat steeper than and shifted to the left of the relation reportedfor hog carotid artery treated 12-16 h in cool 50% glycerol, which developeda maximal tension of ^-0.4 N/cm2 (Mrwa et al ., 1974) .

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Dependence of Tension Development on [Mg-ATP]Fig. 3 shows the average dependence of tension development on externallyprovided Mg-ATP at pCa ^-6.0 measured in eight artery segments from fourarteries . It is interesting to note that isometric tension is maximized by 2 mMMg-ATP, which is about the concentration of high-energy phosphates inintact artery (cf. Butler and Davies, 1980). Below 1 mM external Mg-ATP,tension falls off sharply with decreasing ATP availability . Unlike the force[Ca") dependence, which represents some equilibrium-binding relation, theforce [ATP] relation results from a diffusion-limited reaction rate . Below 2

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FIGURE 2. Active isometric tension developed at various activating free [Ca")is expressed relative to the maximum isometric tension observed in each segmentand plotted against pCa. The data from nine artery segments were grouped andaveraged ; standard error is shown for the three major regions of the data . pKand Nare the parameters of the functional fit shown by the solid line .

mM Mg-ATP, the diffusion rate of ATP into the tissue becomes limiting tothe actomyosin ATPase. This is directly illustrated in Fig. 4, which shows thatunder these conditions, total ATPase falls off directly with tension when ATPis the limiting substrate (A). The response of measured ATPase to external[Mg-ATP] is similar to that of the isometric tension (B) . With [ATP] greaterthan 2 mM, however, isometric tension remains maximized or declines slightlywhile ATPase continues to increase somewhat, perhaps indicating the contri-bution to total ATPase of non-actomyosin ATPases. Alternatively, increasingATPase at constant maximum tension could reflect an increased rate of cross-

FIGURE 3.

Active isometric tension at various external Mg-ATP concentrationsand pCa 6.0 is expressed relative to the tension observed in each artery segmentat 2 mM [Mg-ATP]..The data from eight artery segments have been averagedat each [ATP]. (usually four to six measurements) ; standard deviation is shown .For Mg-ATP concentrations of <5 mM, some MgC12 was added . With theseadditions, between 0 and 5 mM [Mg-ATP], free [Mg"] is calculated to increasefrom 0.28 to 0.43mM.

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THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 " 1952

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FIGURE 4.

The measured tissue ATPase at pCa ^"6.0 in three skinned arterysegments is normalized to the maximum ATPase observed in each arterysegment (^-1.2 Amol ATP/min " g wet artery) and plotted against the externalMg-ATP concentration (B) and the relative isometric force developed at variousexternal Mg-ATP concentrations (A) .

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Tension Development of Vascular Smooth Muscle

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bridge cycling, although the average number of attached cross-bridges (andtherefore tension) is not greatly altered. A similar dissociation of ATPase andtension has been reported for chemically skinned cardiac muscle fibers (Herziget al ., 1981) .

Diffusion MeasurementsThe diffusion rate of [3H]ATP into the preparation was measured directlyand compared with the time-course of tension development under the sameconditions . As detailed in Methods, ATP-free segments were first equilibratedwith activating concentrations of Ca++ so that equilibrium Ca' bindingcould be reached without tension development . This ATP-free Ca" incuba-tion was found to reduce subsequent tension development upon addition ofATP, relative to activation in the opposite order. As shown in Fig. 5, uponaddition of labeled 2 mM Mg-ATP, tension development and ATP permea-tion throughout the tissue rise with similar time-courses (as indicated by thehalf-times to saturation) . Average tissue [ATP] is expressed as counts per gramwet artery segment, while bath [ATP] is counts per cubic centimeter . Inseparate experiments, the water content of hog carotid arteries was estimatedfrom the ratio of dry weight to wet weight . The water content averaged 0.76(± 0.03 SD, n = 21) of the total weight, in agreement with the observationthat ATP equilibrates in 5 min with ^-75% of the total artery volume ascomputed from segment weight (that is, dry weight excludes -25% of thetissue volume). As an approximation, using the diffusion equation for a planeof infinite extent (cf. Crank, 1956), the data obtained are fit by the solid linewith a diffusion constant of 1.4 X 10-s cm2/s, although the fit is not verysensitive to the exact number (as illustrated by the marks at 1 and 3.5 min,which show the range of the fit for DATP between 1 and 2 X 10-s cm2/s) .Even though tissue segments were lightly blotted with filter paper to remove

adherent labeled solution before counting, the data appear to start at arelatively high value, which suggests that about one-third of the availablewater space equilibrates almost instantaneously on the time scale of theseexperiments. The segments for these experiments were purposely cut thick sothat permeation would be slow, thus facilitating the measurements . Recalling,however, the rectangular cross-sectional profile of the segments (0.4 X 0.8 mmin these experiments), a simple calculation shows that surface ATPpenetrationto a depth of only 40 Am could account for this extent of rapid equilibration .This is also perhaps indicative that by cutting off artery segments, surfacedisruption becomes important .

In several experiments, the equilibration with labeled inulin wasdeterminedfor the freeze-glycerinated artery. In agreement with the protein elutionexperiments, which suggest that high molecular weight material is retainedby the skinned preparation, labeled inulin in the artery segment after 15 minrose to a value of 27% (± 3 SD, n = 3) of the bathing solution concentration ;a value that, when taken relative to the total water space (75%), is slightly lessthan that estimated for the extracellular space in hog carotid artery (Murphy

446

et al ., 1974) . Whereas materials of molecular weight on the order of 500 (Mg-ATP and Ca-EGTA, for example) penetrate into the smooth muscle cellsquite readily, inulin with amolecularweight of^5,000 appears to be excluded .This extracellular space could also play some part in the very rapid partialequilibration with labeled ATP.

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THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 " 1982

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FIGURE 5.

Top: the tension development upon addition of 2 mM Mg-ATP to18 skinned artery segments prepared from a single artery is shown . The pCawas 6.0 . Absolute tension development, which was impaired by this procedure,is shown at various times and expressed in gram weight (1 gwt = 0.01 N) .Bottom : the counts per unit volume from [H]ATP in the tissue relative tocounts per unit volume in the incubating solution is shown as a function of timefor the same tissue samples. The solid line shown is the theoretical fit startingfrom the high initial value, which is discussed in the text .

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Dependence of Tension Development Rates on Ca++ and ATPIt was noted in the above experiments that after pre-equilibration withactivating Ca++ in the absence ofATP, tension develops rapidly upon additionof Mg-ATP (time to half-maximal tension development, to.5, was 69 s at 2mM external Mg-ATP). Similar experiments in which tissues remained equil-ibrated with 5 mM Mg-ATP and in which EGTA was abruptly replaced byequimolar Ca-EGTA (pCa 6 .0-6.3 to effect activation) led to very muchslower rates of isometric tension development (to.5 typically 250 s) after asubstantial delay in the onset of activation . Ashley and Moisescu (1973) havedeveloped a technique for reducing the time to produce a step-change ininternal free [Ca"] in skinned skeletal muscle fibers . By first incubatingrelaxed tissues in very low EGTA solutions to reduce Ca++-buffering capacityand then "jumping" to very highly buffered Ca-EGTA-activating solutions(50 mM), changes of internal [Ca++ ] could be produced within 200 ms infibers ^-0.05 mm in diameter. Although the smooth muscle segments used hereare three to five times thicker and the ionic strength considerations limitedthe high Ca'-buffering capacity of the activating solutions to <20 mM, themuch slower time-course of smooth muscle contraction nonetheless permitsthis method to be used to make the Ca` diffusion time short compared withthe half-time of isometric tension development. Assuming that diffusion timegoes as the square of the thickness, we estimate that the internal [Ca++] stepoccurs in <5 s, while to .5 for isometric tension development in the intact arteryactivated with high K+ and histamine averages 34 s (± 8 SD, n = 9) .

Calling "jump intensity" the ratio of [Ca-EGTA] in the activating solutionto [EGTA] in the relaxing solution, it was found for the freeze-glycerinatedartery segments that increasing jump intensity led to progressively faster ratesof isometric tension development (Fig . 6), as did increasing the external [ATP]in experiments with skinned artery segments pre-equilibrated with activating[Ca++], i.e ., "ATPjumps." As shown in Table I, increasing thejump intensityprogressively increased the rate of isometric tension development until thetime-course of tension development saturated at essentially that found for theintact artery supramaximally activated . To allow for possible changes in theshapeof the tension-time relation, two characteristic time constants for tensiondevelopment have been tabulated in Table I (times to 50 and 90% of finaltension) . Further increases in Ca++ jump intensity did not cause the rate oftension development to become faster than that observed in the intact arterypreparation. A similar progression of tension development rate was observedfor ATP jumps.Having found Ca++-activating conditions that maximized the rate of

isometric tension development, the effect of varying activating free [Ca++ ] wasexamined . The results are illustrated in Fig. 7 for a series of "Ca jumps" tovarious free Ca` concentrations . Over the full range of activating [Ca"], theisometric contractions proceed with atime-course that is essentially unaffectedby [Ca"]. This is shown in the lower panel, in which the time-courses of thecontractions at pCa 6.1 and 7.1 have been normalized to the final tensionattained at each pCa. The time-courses virtually superimpose (the other

448 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 " 1982

isometric contractions, which lie between the two shown, have been deletedfor clarity), differing by only ^'25% in the initial steady rate of isometrictension development for a 10-fold change in activating [Ca"] .

TABLE I

100:1 Ca Jump

5mMATP Jump

1 : 1 Ca Jump

8 minFIGURE 6 .

Force-time records for isometric contractions in three skinned arterysegments from the same artery, aligned at the small cup-change artifact, areshown. The slowest (1 :1 Ca jump) shows tension development when relaxing 5mM EGTA is replaced by activating (5 mM EGTA + 3 mM CaC12) . The delayin tension generation onset (^"68 s) reflects the slow increase in intracellular free[Ca"] under these conditions. By decreasing the relaxing [EGTA] to 0.1 MMand increasing activating-Ca" buffering capacity to (10 mM EGTA + 6 mMCaC12), tension develops much more rapidly and the delay is abolished (100:1Ca jump) . The ATP jump (which is performed against no internal ATP-buffering capacity) is as rapid as the 100 :1 Ca jump. The "bump" in the ATPjump tension record was a consistent feature at 2 and 5 mM [ATP], butdisappeared at 10 mM [ATP] . The final tension attained by the 1 :1 Ca jump isshown by the small bar above "ATP."

ISOMETRIC TENSION DEVELOPMENT RATES IN HOG CAROTID ARTERY

Timeconstant

Skinned artery

Intact arteryCa jump intensity

K+ + histamine

s

1:1

10:1 100:1 200:110 .6 250±10 102±16 34±4 3618 34±8to .9 675±147 347±37 84±12 91±8 92±28

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SmM

10 MMto.s 69±8

56±3 34±3

34±8to.9 188±21 104±12 93±17

92±28

to.e and to.s are the times in seconds for isometric tension to reach 50 and 90% of final tension, respectively .Errors shown are standard deviations measured from three to four experiments for each value, rounded offto the whole second . Data for the intact artery are from nine experiments .

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AP. max = 1.13 Kg/cm2

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Top: the time-courses of five isometric contractions by Ca jump atvarious free [Ca"] accumulated from two skinned segments from a single arteryare shown. The jump intensity (100:1) is sufficient to maximize the rate oftension development (cf. Table I) . Force shown in milligram weight (1 mg =10-5 N; 1 kg = 10 N) . Records were aligned at the small cup-change artifact .Bottom : the two extreme traces above are normalized to the final tensionsattained ; other traces lie between. Initial rates of tension development have beentaken from the early linear phase.

From the measurements of total tissue ATPase (- 1 .2 ,umol/min-gram arterywhen maximally activated) and the ATP diffusion constant (-1 .4 X 10-scm2/S), the ATP concentration profile throughout the thickness ofthe skinned

450 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 79 - 1982

artery segment can be estimated from the steady-state reaction-diffusionequation for an infinite plane sheet (cf. Crank, 1956) . At 2 mM Mg-ATP inthe bathing solution, the concentration of ATP in the tissue is apparentlyadequate to support maximum tension generation, since increasing [ATP]odoes not increase tension (Fig . 3) . From the values presented above, the centralATP concentration for artery segments 0.3 mm thick would be on the orderof 0.5 mM. Below this minimum internal [ATP], tension apparently cannotbe maximally maintained . For comparison, Murphy (1969) has found foractomyosin isolated from hog carotid artery that actomyosin ATPase declinesby only -15% when [ATP] is reduced from 1 to 0.5 mM, but by -70% when[ATP] is decreased from 0.5 to 0.1 mM . Taking 0.5 mM ATP as the minimumconcentration needed to support full tension and using the estimated diffu-sion constant, then for artery segments given ample time to equilibratewith activating [Ca"] in ATP-free solution, the abrupt addition of 10 mM[ATP]o would cause internal [ATP] to exceed 0.5 mM everywhere in the tissuein ^"30 s (±10) . Whereas [ATP] adequate to support maximum tensiongeneration is therefore available in ---30 s, isometric tension under the sameconditions requires -90 s to approach maximum tension. ATP availabilitydoes not, apparently, limit the rate of isometric tension development.

It was shown above that the rate of isometric tension development inskinned hog carotid artery can be made identical to the rate of isometrictension development in the intact artery if the methods of "Ca jump" areused . Under these conditions, making intracellular Ca" rapidly available foractivation (within ^-5 s) does not cause the skinned preparation to developtension more rapidly than does the intact artery activated hormonally and bymembrane depolarization (the half-time is "̂35 s in both cases) . Furthermore,the rate of tension development relative to the final tension attained is notaffected by the activating free [Ca"] (Fig . 7) . These observations indicatethat the rate-limiting process in isometric tension development is not directlydependent on Ca". That is, processes related to the sudden appearance ofactivator-Ca" (such as binding) are rapid compared with subsequent stepssuch as myosin light chain phosphorylation and actomyosin cross-bridgeformation . Since the observed rates of isometric tension development in theskinned preparation are identical to those found in the intact preparation, itseems fair to extrapolate this conclusion to intact arterial smooth muscle . Afteractivation of physiologically intact vascular smooth muscle, the rate at whichintracellular free [Ca"] increases is not rate-limiting for tension development.

The author wishes to thank Prof J. C. Ruegg who supported this work, and especially FrauDoris Eubler who assisted in all experiments and Dr . P. J. Griffiths who suggested the use of"Cajumps."

Receivedfor publication 21 August 1981 and in revisedform 5 November 1981.

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